Compositions and methods to prevent cancer with cupredoxins

ABSTRACT

The present invention relates to compositions comprising peptides that may be variants, derivatives and structural equivalents of cupredoxins that inhibit the development of premalignant lesions in mammalian cells, tissues and animals. Specifically, these compositions may comprise azurin from  Pseudomonas aeruginosa , and/or the 50-77 residue region of azurin (p28). The present invention further relates to compositions that may comprise cupredoxin(s), and/or variants, derivatives or structural equivalents of cupredoxins, that retain the ability to inhibit the development of premalignant lesions in mammalian cells, tissues or animals. These compositions may be peptides or pharmaceutical compositions, among others. The compositions of the invention may be used to prevent the development of premalignant lesions in mammalian cells, tissues and animals, and thus prevent cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §§119 and 120, toProvisional U.S. Application Ser. No. 60/844,358, filed Sep. 14, 2006;and is a continuation of U.S. patent application Ser. No. 12/617,841,filed Nov. 13, 2009, and is a continuation of U.S. patent applicationSer. No. 11/854,654, filed Sep. 13, 2007, which issued as U.S. Pat. No.7,618,939 on Nov. 17, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 11/244,105, filed Oct. 6, 2005, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/616,782,filed Oct. 7, 2004, and U.S. Provisional Patent Application Ser. No.60/680,500, filed May 13, 2005; and is a continuation-in-part of U.S.patent application Ser. No. 10/720,603, filed Nov. 24, 2003, whichissued as U.S. Pat. No. 7,491,394 on Feb. 17, 2009, and which claimspriority to U.S. Provisional Patent Application Ser. No. 60/414,550,filed Aug. 15, 2003, and which is a continuation-in-part of U.S. patentapplication Ser. No. 10/047,710, filed Jan. 15, 2002, which issued asU.S. Pat. No. 7,084,105 on Aug. 1, 2006, and which claims priority toU.S. Provisional Patent Application Ser. No. 60/269,133, filed Feb. 15,2001. The entire content of these prior applications is fullyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions comprising variants,derivatives and structural equivalents of cupredoxins that inhibit thedevelopment of premalignant lesions in mammalian cells, tissues andanimals. The invention also relates to the use of cupredoxins, andvariants, derivatives and structurally equivalents of cupredoxins, aschemopreventive agents in mammals to inhibit the development ofpremalignant lesions, and ultimately cancer.

BACKGROUND

Cancer chemoprevention is the use of natural, synthetic or biologicchemical agents to reverse, suppress, or prevent carcinogenicprogression to invasive cancer. Recent clinical trials in preventingcancer in high-risk populations suggest that chemopreventive therapy isa realistic treatment for high-risk patients. Chemopreventive therapy isbased on the concepts of multifocal field carcinogenesis and multistepcarcinogenesis. In field carcinogenesis, generalized carcinogen exposurethroughout the tissue field results in diffuse epithelial injury intissue and clonal proliferation of the mutated cells. These geneticmutations throughout the field increase the likelihood that one or morepremalignant or malignant lesions may develop in the field. Multistepcarcinogenesis in the stepwise accumulation of these genetic andphenotypic alterations. Arresting one or more steps in the multistepcancinogenesis may impede or prevent the development of cancer. Seegenerally Tsao et al., CA Cancer J Clin 54:150-180 (2004).

Azurin, and other cupredoxins, are cytotoxic specifically towards cancercells. Azurin induces apoptosis in J774 lung cancer cells. Yamada etal., PNAS 99(22):14098-14103 (2002). On entry into J774 lung cancercells, azurin localizes in the cytosol and nuclear fractions, and formsa complex with tumor suppressor protein p53, thereby stabilizing it andenhancing its intracellular level. Id. The induction of azurin-mediatedapoptosis is not limited to J774 cells. Azurin can also enter cancercells such as human melanoma UISO-Mel-2 or human breast cancer MCF-7cells. Yamada et al., Infect Immun. 70:7054-7062 (2002); Punj et al.,Oncogene. 23:2367-2378 (2004). In both cases, azurin allowed theelevation of the intracellular p53 levels, leading to enhanced Baxformation and induction of apoptosis in such cells. Most interestingly,intraperitoneal injection of azurin in nude mice harboring xenograftedMel-2 or MCF-7 human cancers led to statistically significant regressionof such cancers. Id.

The mouse mammary gland organ culture (MMOC) assay may be used toevaluate the inhibitory effects of potential chemopreventive agents onboth hormone-induced structural differentiation of mammary glands and onthe development of DMBA-induced preneoplastic hyperplastic alveolarnodule-like lesions in the gland. Mammary glands from young, virginanimals, when incubated for 6 days in the presence of insulin(I)+prolactin (P)+aldosterone (A), can differentiate into fully-grownglands. These glands morphologically resemble the glands obtained frompregnant mice. Aldosterone can be replaced by estrogen (E)+progesterone(Pg) Inclusion of hydrocortisone (H) to the medium stimulates thefunctional differentiation of the mammary glands. Mehta and Banerjee,Acta Endocrinol. 80:501 (1975); Mehta and Moon, Breast Cancer: Treatmentand Prognosis 300, 300 (Basil A Stoll ed., Blackwell Press 1986). Thus,the hormone-induced structural and functional differentiation, observedin this culture system, mimics the responses to hormones observed duringvarious physiological stages of the animal.

Mice exhibit a distinct preneoplastic stage prior to cancer formation inMMOC. Such preneoplastic lesions in C3H mice are induced by murinemammary tumor virus or in BALB/c mice by DMBA. Exposure of the glands to2 μg/ml DMBA between days 3 and 4 of growth phases followed byregression of the glands for 2-3 weeks in the medium containing onlyinsulin, results in the formation of mammary alveolar lesions (MAL).Hawthorne et al., Pharmaceutical Biology 40:70-74 (2002); Mehta et al.,Methods in Cell Science 19:19-24 (1997). Furthermore, transplantation ofepithelial cells, prepared from glands containing the DMBA-inducedmammary lesions, into syngeneic host resulted in the development ofmammary adenocarcinoma. Telang et al., PNAS 76:5886-5890 (1979).Pathologically, these tumors were similar to those observed in vivo whenmice of the same strain are administered DMBA. Id.

DMBA-induced mammary lesion formation in MMOC can be inhibited by avariety of classes of chemopreventive agents such as retinoids. Theseagents include chemopreventive agents derived from the natural productssuch as brassinin and resveretrol, thiols, antioxidants, inhibitors ofornithine decarboxylase such as OFMO and deguelin, inhibitors ofprostaglandin synthesis, Ca regulators, etc. Jang et al., Science275:218-220 (1997); Mehta, Eur. J. Cancer 36:1275-1282 (2000); Metha etal., J. Natl. Cancer Inst. 89:212-219 (1997). These studies clearlydemonstrate that this organ culture system offers a unique model todetermine the effectiveness of compounds against mammary carcinogenesis.The results can be expected to closely correlate to the inhibitionobtained by in vivo administration of such compounds.

The MMOC may also be induced to form mammary ductal lesions (MDL). TheMDL can be induced if estrogen and progesterone instead of aldosteroneand hydrocortisone are included in the medium. The alveolar structuresin the presence of ovarian steroids are very small but the intraductallesions are observed in histopathological sections. Mehta et al., J.Natl. Cancer Inst. 93:1103-1106 (2001). The antiestrogens, whichselectively work on ovarian hormone dependent ER+breast cancers such astamoxifen, inhibited MDL formation and not MAL. Thus, this modifiedculture model in addition to conventional MAL induction protocol now canbe used to evaluate effects of chemopreventive agents on both MAL andMOL.

What is needed is a chemopreventive agent that inhibit the developmentof premalignant lesions. Such a chemopreventive agent should be able toeither prevent the initial development of premalignant lesions, inducecell death in premalignant lesions that form, and/or prevent thedevelopment of premalignant lesions into malignant lesions. Suchchemopreventive agents would have great utility in treating, inparticular, patients who are at a high risk of developing cancer, due toeither the presence of high-risk features, the presence of pre-malignantlesions, or the previous of cancer or premalignant lesions.

SUMMARY OF THE EMBODIMENTS

The present invention relates to compositions comprising peptides thatmay be variants, derivatives and structural equivalents of cupredoxinsthat inhibit the development of premalignant lesions in mammalian cells,tissues and animals. Specifically, these compositions may compriseazurin from Pseudomonas aeruginosa, and/or the 50-77 residue region ofazurin (p28). The present invention further relates to compositions thatmay comprise cupredoxin(s), and/or variants, derivatives or structuralequivalents of cupredoxins, that retain the ability to inhibit thedevelopment of premalignant lesions in mammalian cells, tissues oranimals. These compositions may be isolated peptides or pharmaceuticalcompositions, among others. The compositions of the invention may beused in methods to prevent the development of cancer in mammalianpatients.

One aspect of the invention are isolated peptides that may be a variant,derivative or structural equivalent of a cupredoxin; and may inhibit thedevelopment of premalignant lesions in mammalian tissue. The cupredoxinmay be azurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin,stellacyanin and cucumber basic protein, and specifically may be azurin.The cupredoxin may be from an organism such as Pseudomonas aeruginosa,Alcaligenes faecalis, Ulva pertussis, Achromobacter xylosoxidan,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis,Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis,Xylella fastidiosa and Vibrio parahaemolyticus, and specifically may bePseudomonas aeruginosa. In some embodiments, the peptide may be part ofSEQ ID NOS: 1, 3-19, or has at least 80% amino acid sequence identity toSEQ ID NOS: 1, 3-19.

In some embodiments, the isolated peptide may be a truncation of acupredoxin. The isolated peptide may be more than about 10 residues andnot more than about 100 residues. The isolated peptide may comprise, oralternatively consist of, Pseudomonas aeruginosa azurin residues 50-77,Pseudomonas aeruginosa azurin residues 50-67, Pseudomonas aeruginosaazurin residues 36-88, or SEQ ID NOS: 20-24.

Another aspect of the invention is a pharmaceutical composition that maycomprise at least one, or two, cupredoxins or isolated peptides of theinvention in a pharmaceutically acceptable carrier. The pharmaceuticalcomposition may be formulated for intravenous administration. In someembodiments, the cupredoxin in the pharmaceutical composition may befrom an organism such as Pseudomonas aeruginosa, Ulva pertussis,Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetellabronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseriagonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa and Vibrio parahaemolyticus, and specifically may be fromPseudomonas aeruginosa. The cupredoxin may be SEQ ID NOS: 1, 3-19.

Another aspect of the invention is a method to treat a mammalian patientby administering to the patient a therapeutically effective amount ofthe pharmaceutical composition of the invention. The patient may behuman, and may be at a higher risk to develop cancer than the generalpopulation. In some embodiments, the cancer may be melanoma, breast,pancreas, glioblastoma, astrocytoma, lung, colorectal, neck and head,bladder, prostate, skin, or cervical cancer. In some embodiments, thepatient may have at least one high risk feature, premalignant lesions orhave been cured of cancer or premalignant lesions.

The pharmaceutical composition may be administered by intravenousinjection, intramuscular injection, subcutaneous injection, inhalation,topical administration, transdermal patch, suppository, vitreousinjection or oral, and specifically may be administered by intravenousinjection. The pharmaceutical composition may be co-administered with atleast one other chemopreventive drug, and specifically at about the sametime as another chemopreventive drug.

Another aspect of the invention is a kit comprising the pharmaceuticalcomposition of the invention in a vial. The kit may be designed forintravenous administration.

Another aspect of the invention is a method to study the development ofcancer comprising contacting mammalian cells with a cupredoxin orpeptide of the invention and measuring the development of premalignantand malignant cells. In some embodiments, the cells may be human and/ormammary cells. In some embodiments, the cells are induced to developpremalignant lesions.

Another aspect of the invention is an expression vector, which encodes apeptide of the invention.

These and other aspects, advantages, and features of the invention willbecome apparent from the following figures and detailed description ofthe specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1. Amino acid sequence of azurin from Pseudomonas aeruginosa.

SEQ ID NO: 2. Amino acid sequence of p28, Pseudomonas aeruginosa azurinresidues 50-77.

SEQ ID NO: 3. Amino acid sequence of plastocyanin from Phormidiumlaminosum.

SEQ ID NO: 4. Amino acid sequence of rusticyanin from Thiobacillusferrooxidans.

SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobactercycloclastes.

SEQ ID NO: 6. Amino acid sequence of azurin from Alcaligenes faecalis.

SEQ ID NO: 7. Amino acid sequence of azurin from Achromobacterxylosoxidans ssp. denitrificans I.

SEQ ID NO: 8. Amino acid sequence of azurin from Bordetellabronchiseptica.

SEQ ID NO: 9. Amino acid sequence of azurin from Methylomonas sp. J.

SEQ ID NO: 10. Amino acid sequence of azurin from Neisseria meningitidisZ2491.

SEQ ID NO: 11. Amino acid sequence of azurin from Pseudomonasfluorescen.

SEQ ID NO: 12. Amino acid sequence of azurin from Pseudomonaschlororaphis.

SEQ ID NO: 13. Amino acid sequence of azurin from Xylella fastidiosa9a5c.

SEQ ID NO: 14. Amino acid sequence of stellacyanin from Cucumis sativus.

SEQ ID NO: 15. Amino acid sequence of auracyanin A from Chloroflexusaurantiacus.

SEQ ID NO: 16. Amino acid sequence of auracyanin B from Chloroflexusaurantiacus.

SEQ ID NO: 17. Amino acid sequence of cucumber basic protein fromCucumis sativus.

SEQ ID NO: 18. Amino acid sequence of Laz from Neisseria gonorrhoeaeF62.

SEQ ID NO: 19. Amino acid sequence of the azurin from Vibrioparahaemolyticus.

SEQ ID NO: 20. Amino acid sequence of amino acids 57 to 89 of auracyaninB of Chloroflexus aurantiacus.

SEQ ID NO: 21. Amino acid sequence of amino acids 51-77 of Pseudomonassyringae azurin.

SEQ ID NO: 22. Amino acid sequence of amino acids 89-115 of Neisseriameningitidis Laz.

SEQ ID NO: 23. Amino acid sequence of amino acids 52-78 of Vibrioparahaemolyticus azurin.

SEQ ID NO: 24. Amino acid sequence of amino acids 51-77 of Bordetellabronchiseptica azurin.

SEQ ID NO: 25. Amino acid sequence of amino acids 50-67 of Pseudomonasaeruginosa azurin.

SEQ ID NO: 26. Amino acid sequence of amino acids 36-88 of Pseudomonasaeruginosa azurin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FIG. 1 depicts photographs of all of the glands evaluated forthe efficacy of p28 and azurin. FIG. 1A shows a representativephotograph of alveolar lesions in a DMBA-treated gland and itscomparison with a gland that was treated with DMBA along with achemopreventive agent. FIGS. 1B-1G show representative photographs ofthe effects of p28 on the development of alveolar lesions.

FIG. 2. FIG. 2 depicts a graph showing the efficacy of p28 againstDMBA-induced mammary alveolar lesions.

FIG. 3. FIG. 3 depicts photographs of representative sections of ductallesions and effect of p28.

FIG. 4. FIG. 4 depicts a graph showing the efficacy of p28 againstDMBA-induced ductal lesions

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “cell” includes either the singular or theplural of the term, unless specifically described as a “single cell.”

As used herein, the terms “polypeptide,” “peptide,” and “protein” areused interchangeably to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid. The terms also apply to naturally occurring aminoacid polymers. The terms “polypeptide,” “peptide,” and “protein” arealso inclusive of modifications including, but not limited to,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation. It will beappreciated that polypeptides are not always entirely linear. Forinstance, polypeptides may be branched as a result of ubiquitination andthey may be circular (with or without branching), generally as a resultof post-translation events, including natural processing event andevents brought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods aswell.

As used herein, the term “pharmacologic activity” means the effect of adrug or other chemical on a biological system. The effect of chemicalmay be beneficial (therapeutic) or harmful (toxic). The pure chemicalsor mixtures may be of natural origin (plant, animal, or mineral) or maybe synthetic compounds.

As used herein, the term “premalignant” means precancerous, or beforeabnormal cells divide without control.

As used herein, the term “lesion” means an area of abnormal tissue.

As used herein, the term “pathological condition” includes anatomic andphysiological deviations from the normal that constitute an impairmentof the normal state of the living animal or one of its parts, thatinterrupts or modifies the performance of the bodily functions, and is aresponse to various factors (as malnutrition, industrial hazards, orclimate), to specific infective agents (as worms, parasitic protozoa,bacteria, or viruses), to inherent defects of the organism (as geneticanomalies), or to combinations of these factors.

As used herein, the term “condition” includes anatomic and physiologicaldeviations from the normal that constitute an impairment of the normalstate of the living animal or one of its parts, that interrupts ormodifies the performance of the bodily functions.

As used herein, the term “suffering from” includes presently exhibitingthe symptoms of a pathological condition, having a pathologicalcondition even without observable symptoms, in recovery from apathological condition, or recovered from a pathological condition.

As used herein, the term “chemoprevention” is the use of drugs,vitamins, or other agents to try to reduce the risk of, or delay thedevelopment or recurrence of, cancer.

A used herein, the term “treatment” includes preventing, lowering,stopping, or reversing the progression or severity of the condition orsymptoms associated with a condition being treated. As such, the term“treatment” includes medical, therapeutic, and/or prophylacticadministration, as appropriate. Treatment may also include preventing orlessening the development of a condition, such as cancer.

As used herein, the term “inhibit cell growth” means the slowing orceasing of cell division and/or cell expansion. This term also includesthe inhibition of cell development or increases in cell death.

A “therapeutically effective amount” is an amount effective to prevent,lower, stop or reverse the development of, or to partially or totallyalleviate the existing symptoms of a particular condition for which thesubject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art.

The term “substantially pure,” as used herein, when used to modify aprotein or other cellular product of the invention, refers to, forexample, a protein isolated from the growth medium or cellular contents,in a form substantially free of, or unadulterated by, other proteinsand/or other compounds. The term “substantially pure” refers to a factorin an amount of at least about 75%, by dry weight, of isolated fraction,or at least “75% substantially pure.” More specifically, the term“substantially pure” refers to a compound of at least about 85%, by dryweight, of isolated fraction, or at least “85% substantially pure.” Mostspecifically, the term “substantially pure” refers to a compound of atleast about 95%, by dry weight, of isolated fraction, or at least “95%substantially pure.” The term “substantially pure” may also be used tomodify a synthetically-made protein or compound of the invention, where,for example, the synthetic protein is isolated from the reagents andby-products of the synthesis reaction(s).

The term “pharmaceutical grade,” as used herein, when referring to apeptide or compound of the invention, is a peptide or compound that isisolated substantially or essentially from components which normallyaccompany the material as it is found in its natural state, includingsynthesis reagents and by-products, and substantially or essentiallyisolated from components that would impair its use as a pharmaceutical.For example, a “pharmaceutical grade” peptide may be isolated from anycarcinogen. In some instances, “pharmaceutical grade” may be modified bythe intended method of administration, such as “intravenouspharmaceutical grade,” in order to specify a peptide or compound that issubstantially or essentially isolated from any substance that wouldrender the composition unsuitable for intravenous administration to apatient. For example, an “intravenous pharmaceutical grade” peptide maybe isolated from detergents, such as SDS, and anti-bacterial agents,such as azide.

The terms “isolated,” “purified” or “biologically pure” refer tomaterial which is substantially or essentially free from componentswhich normally accompany the material as it is found in its nativestate. Thus, isolated peptides in accordance with the inventionpreferably do not contain materials normally associated with thepeptides in their in situ environment. An “isolated” region of apolypeptide refers to a region that does not include the whole sequenceof the polypeptide from which the region was derived. An “isolated”nucleic acid, protein, or respective fragment thereof has beensubstantially removed from its in vivo environment so that it may bemanipulated by the skilled artisan, such as but not limited to,nucleotide sequencing, restriction digestion, site-directed mutagenesis,and subcloning into expression vectors for a nucleic acid fragment aswell as obtaining the protein or protein fragment in substantially purequantities.

The term “variant” as used herein with respect to a peptide, refers toamino acid sequence variants which may have amino acids replaced,deleted, or inserted as compared to the wild-type polypeptide. Variantsmay be truncations of the wild-type peptide. A “deletion” is the removalof one or more amino acids from within the polypeptide, which a“truncation” is the removal of one or more amino acids from one or bothends of the polypeptide. Thus, a variant peptide may be made bymanipulation of genes encoding the polypeptide. A variant may be made byaltering the basic composition or characteristics of the polypeptide,but not at least some of its pharmacologic activities. For example, a“variant” of azurin can be a mutated azurin that retains its ability toinhibit the development of premalignant mammalian cells. In some cases,a variant peptide is synthesized with non-natural amino acids, such asε-(3,5-dinitrobenzoyl)-Lys residues. Ghadiri & Fernholz, J. Am. Chem.Soc., 112:9633-9635 (1990). In some embodiments, the variant has notmore than 20 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 15 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 10 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 6 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 5 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof. In some embodiments, the variant hasnot more than 3 amino acids replaced, deleted or inserted compared towild-type peptide or part thereof.

The term “amino acid,” as used herein, means an amino acid moiety thatcomprises any naturally-occurring or non-naturally occurring orsynthetic amino acid residue, i.e., any moiety comprising at least onecarboxyl and at least one amino residue directly linked by one, twothree or more carbon atoms, typically one (a) carbon atom.

The term “derivative” as used herein with respect to a peptide refers toa peptide that is derived from the subject peptide. A derivationincludes chemical modifications of the peptide such that the peptidestill retains some of its fundamental activities. For example, a“derivative” of azurin can, for example, be a chemically modified azurinthat retains its ability to inhibit angiogenesis in mammalian cells.Chemical modifications of interest include, but are not limited to,amidation, acetylation, sulfation, polyethylene glycol (PEG)modification, phosphorylation or glycosylation of the peptide. Inaddition, a derivative peptide may be a fusion of a polypeptide orfragment thereof to a chemical compound, such as but not limited to,another peptide, drug molecule or other therapeutic or pharmaceuticalagent or a detectable probe.

The term “percent (%) amino acid sequence identity” is defined as thepercentage of amino acid residues in a polypeptide that are identicalwith amino acid residues in a candidate sequence when the two sequencesare aligned. To determine % amino acid identity, sequences are alignedand if necessary, gaps are introduced to achieve the maximum % sequenceidentity; conservative substitutions are not considered as part of thesequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Oftenpublicly available computer software such as BLAST, BLAST2, ALIGN2 orMegalign (DNASTAR) software is used to align peptide sequences. In aspecific embodiment, Blastp (available from the National Center forBiotechnology Information, Bethesda Md.) is used using the defaultparameters of long complexity filter, expect 10, word size 3, existence11 and extension 1.

When amino acid sequences are aligned, the % amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain % amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:

% amino acid sequence identity=X/Y*100

where

-   -   X is the number of amino acid residues scored as identical        matches by the sequence alignment program's or algorithm's        alignment of A and B and    -   Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length ofamino acid sequence B, the % amino acid sequence identity of A to B willnot equal the % amino acid sequence identity of B to A. When comparinglonger sequences to shorter sequences, the shorter sequence will be the“B” sequence. For example, when comparing truncated peptides to thecorresponding wild-type polypeptide, the truncated peptide will be the“B” sequence.

General

The present invention provides compositions comprising cupredoxin, andvariants, derivatives and structural equivalents of cupredoxins, andmethods to prevent the development of cancer in mammals. The inventionalso provides to variants, derivatives and structural equivalents ofcupredoxin that retain the ability to prevent the development of canceror the re-occurrence of cancer in mammals. Most particularly, theinvention provides compositions comprising Pseudomonas aeruginosaazurin, variants, derivatives and structural equivalents of azurin, andtheir use to treat patients, and particularly patients at a higher riskof developing cancer than the general population. Finally, the inventionprovides methods to study the development of cancer in mammalian cells,tissues and animals by contacting the cells with a cupredoxin, orvariant, derivative or structural equivalent thereof, before or afterinducing premalignant lesions, and observing the development ofpremalignant and/or malignant cells.

Previously, it was know that a redox protein elaborated by Pseudomonasaerugisnosa, the cupredoxin azurin, selectively enters J774 lung cancercells but not normal cells, and induces apoptosis. Zaborina et al.,Microbiology 146:2521-2530 (2000). Azurin can also selectively enter andkill human melanoma UISO-MeI-2 or human breast cancer MCF-7 cells.Yamada et al., PNAS 99:14098-14103 (2002); Punj et al., Oncogene23:2367-2378 (2004). Azurin from P. aeruginosa preferentially entersJ774 murine reticulum cell sarcoma cells, forms a complex with andstabilizes the tumor suppressor protein p53, enhances the intracellularconcentration of p53, and induces apoptosis. Yamada et al., Infectionand Immunity 70:7054-7062 (2002). Detailed studies of various domains ofthe azurin molecule showed that amino acids 50-77 (p28) (SEQ ID NO: 2)represented a protein transduction domain (PTD) critical forinternalization and subsequent apoptotic activity. Yamada et al., Cell.Microbial. 7:1418-1431 (2005).

It is now known that azurin, and peptides derived from azurin, such asp28, have chemopreventive properties. It is now known that azurin, andp28, prevent to formation of premalignant preneoplastic lesions in mousemammary gland organ culture. In a mouse mammary gland organ culturemodel, azurin at 50 μg/ml was found to inhibit the formation of alveolarlesions by 67%. Likewise, p28 at 25 μg/ml was found to inhibit theformation of alveolar lesions by 67%. See Example 1. Further, azurin at50 μg/ml was found to inhibit the formation of ductal lesions by 79%,and p28 at 25 μg/ml inhibited the formation of ductal lesions by 71%.See Example 1. Confocal microscopy and FAC showed that azurin and p28entered normal murine mammary epithelial cells (MM3MG) and mammarycancer cells (4T1). P28 also entered human umbilical vein endothelialcells (HUVEC) in a temperature, time and concentration dependent mannerand inhibited capillary tube formation of HUVEC plated on Matrigel® in adose dependent manner. It is therefore now known that azurin andvariants of azurin may be used to inhibit the formation of premalignantpreneoplastic lesions, and thus the development of cancer, andspecifically breast cancer, in mammalian patients.

Due to the high degree of structural similarity between cupredoxins, itis likely that other cupredoxins will inhibit the formation ofpremalignant lesions in mammals as well as azurin. Such cupredoxins maybe found in, for example, bacteria or plants. Several cupredoxins areknown to have pharmacokinetic activities similar to those of azurin fromPseudomonas aeruginosa. For example, rusticyanin from Thiobacillusferrooxidans can also enter macrophages and induce apoptosis. Yamada etal., Cell Cycle 3:1182-1187 (2004); Yamada et al., Cell. Micro.7:1418-1431 (2005). Plastocyanin from Phormidium laminosum andpseudoazurin form Achromobacter cycloclastes also are cytotoxic towardsmacrophages. U.S. Pat. Pub. No. 20060040269, published Feb. 23, 2006. Itis therefore contemplated that other cupredoxins may be used in thecompositions and methods of the invention. Further, variants,derivatives, and structural equivalents of cupredoxins that retain theability to inhibit the formation of cancer in mammals may also be usedin the compositions and methods of the invention. These variants andderivatives may include, but are not limited to, truncations of acupredoxin, conservative substitutions of amino acids and proteinsmodifications such as PEGylation and all-hydrocarbon stabling ofα-helices.

Compositions of the Invention

The invention provides for peptides that are variants, derivatives orstructural equivalents of cupredoxin that inhibit the development ofpremalignant lesions in mammalian cells, tissues and animals. Theinvention further provides for peptides that are variants, derivativesor structural equivalents of cupredoxin that inhibit the development ofcancer in mammalian cells, tissues and animals. In some embodiments, thepeptide is isolated. In some embodiments, the peptide is substantiallypure or pharmaceutical grade. In other embodiments, the peptide is in acomposition that comprises, or consists essentially of, the peptide. Inanother specific embodiment, the peptide is non-antigenic and does notraise an immune response in a mammal, and more specifically a human. Insome embodiments, the peptide is less that a full-length cupredoxin, andretains some of the pharmacologic activities of the cupredoxins.Specifically, in some embodiments, the peptide may retain the ability toinhibit the development of premalignant lesions in the mouse mammarygland organ culture.

The invention also provides compositions comprising at least one peptidethat is a cupredoxin, or variant, derivative or structural equivalent ofa cupredoxin, specifically in a pharmaceutical composition. In specificembodiments, the pharmaceutical composition is designed for a particularmode of administration, for example, but not limited to, oral,intraperitoneal, or intravenous. Such compositions may be hydrated inwater, or may be dried (such as by lyophilization) for later hydration.Such compositions may be in solvents other than water, such as but notlimited to, alcohol.

Because of the high structural homology between the cupredoxins, it iscontemplated that cupredoxins will have the same chemopreventiveproperties as azurin and p28. In some embodiments, the cupredoxin is,but is not limited to, azurin, pseudoazurin, plastocyanin, rusticyanin,auracyanin, stellacyanin, cucumber basic protein or Laz. In particularlyspecific embodiments, the azurin is derived from Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidans ssp. denitrificans I,Bordetella bronchiseptica, Methylomonas sp., Neisseria meningitidis,Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis,Xylella fastidiosa, Ulva pertussis or Vibrio parahaemolyticus. In a veryspecific embodiment, the azurin is from Pseudomonas aeruginosa. In otherspecific embodiments, the cupredoxin comprises an amino acid sequencethat is SEQ ID NO: 1, 3-19.

The invention provides peptides that are amino acid sequence variantswhich have amino acids replaced, deleted, or inserted as compared to thewild-type cupredoxin. Variants of the invention may be truncations ofthe wild-type cupredoxin. In some embodiments, the peptide of theinvention comprises a region of a cupredoxin that is less that the fulllength wild-type polypeptide. In some embodiments, the peptide of theinvention comprises more than about 10 residues, more than about 15residues or more than about 20 residues of a truncated cupredoxin. Insome embodiments, the peptide comprises not more than about 100residues, not more than about 50 residues, not more than about 40residues, not more than about 30 residues or not more than about 20residues of a truncated cupredoxin. In some embodiments, a cupredoxinhas to the peptide, and more specifically SEQ ID NOS: 1, 3-19 as to thepeptide of the invention, at least about 70% amino acid sequenceidentity, at least about 80% amino acid sequence identity, at leastabout 90% amino acid sequence identity, at least about 95% amino acidsequence identity or at least about 99% amino acid sequence identity.

In specific embodiments, the variant of cupredoxin comprises P.aeruginosa azurin residues 50-77 (p28, SEQ ID NO: 2), azurin residues50-67, or azurin residues 36-88. In other embodiments, the variant ofcupredoxin consists of P. aeruginosa azurin residues 50-77, azurinresidues 50-67, or azurin residues 36-88. In other specific embodiments,the variant consists of the equivalent residues of a cupredoxin otherthat azurin. It is also contemplated that other cupredoxin variants canbe designed that have a similar pharmcologic activity to azurin residues50-77, or azurin residues 36-88. To do this, the subject cupredoxinamino acid sequence will be aligned to the Pseudomonas aeruginosa azurinsequence using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevantresidues located on the P. aeruginosa azurin amino acid sequence, andthe equivalent residues found on the subject cupredoxin sequence, andthe equivalent peptide thus designed.

In one embodiment of the invention, the cupredoxin variant contains atleast amino acids 57 to 89 of auracyanin B of Chloroflexus aurantiacus(SEQ ID NO: 20). In another embodiment of the invention, the cupredoxinvariant contains at least amino acids 51-77 of Pseudomonas syringaeazurin (SEQ ID NO: 21). In another embodiment of the invention, thecupredoxin variant contains at least amino acids 89-115 of Neisseriameningitidis Laz (SEQ ID NO: 22). In another embodiment of theinvention, the cupredoxin variant contains at least amino acids 52-78 ofVibrio parahaemolyticus azurin (SEQ ID NO: 23). In another embodiment ofthe invention, the cupredoxin variant contains at least amino acids51-77 of Bordetella bronchiseptica azurin (SEQ ID NO: 24).

The variants may also include peptides made with synthetic amino acidsnot naturally occurring. For example, non-naturally occurring aminoacids may be integrated into the variant peptide to extend or optimizethe half-life of the composition in the bloodstream. Such variantsinclude, but are not limited to, D,L-peptides (diastereomer), (forexample Futaki et al., J. Biol. Chem. 276(8):5836-40 (2001); Papo etal., Cancer Res. 64(16):5779-86 (2004); Miller et al, Biochem.Pharmacol. 36(1):169-76, (1987); peptides containing unusual amino acids(for example Lee et al., J. Pept. Res. 63(2):69-84 (2004)),olefin-containing non-natural amino acid followed by hydrocarbonstapling (for example Schafineister et al., J. Am. Chem. Soc.122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)),and peptides comprising ε-(3,5-dinitrobenzoyl)-Lys residues.

In other embodiments, the peptide of the invention is a derivative of acupredoxin. The derivatives of cupredoxin are chemical modifications ofthe peptide such that the peptide still retains some of its fundamentalactivities. For example, a “derivative” of azurin can be a chemicallymodified azurin that retains its ability to inhibit the development ofpremalignant lesions in mammalian cells, tissues or animals. Chemicalmodifications of interest include, but are not limited to, hydrocarbonstabling, amidation, acetylation, sulfation, polyethylene glycol (PEG)modification, phosphorylation and glycosylation of the peptide. Inaddition, a derivative peptide maybe a fusion of a cupredoxin, orvariant, derivative or structural equivalent thereof to a chemicalcompound, such as but not limited to, another peptide, drug molecule orother therapeutic or pharmaceutical agent or a detectable probe.Derivatives of interest include chemical modifications by which thehalf-life in the bloodstream of the peptides and compositions of theinvention can be extended or optimized, such as by several methods wellknown to those in the art, including but not limited to, circularizedpeptides (for example Monk et al., BioDrugs 19(4):261-78, (2005);DeFreest et al., J. Pept. Res. 63(5):409-19 (2004)), N- and C-terminalmodifications (for example Labrie et al., Clin. Invest. Med.13(5):275-8, (1990)), and olefin-containing non-natural amino acidfollowed by hydrocarbon stapling (for example Schafineister et al., J.Am. Chem. Soc. 122:5891-5892 (2000); Walenski et al., Science305:1466-1470 (2004)).

In another embodiment, the peptide is a structural equivalent of acupredoxin. Examples of studies that determine significant structuralhomology between cupredoxins and other proteins include Toth et al.(Developmental Cell 1:82-92 (2001)). Specifically, significantstructural homology between a cupredoxin and the structural equivalentmay be determined by using the VAST algorithm. Gibrat et al., Curr OpinStruct Biol 6:377-385 (1996); Madej et al., Proteins 23:356-3690 (1995).In specific embodiments, the VAST p value from a structural comparisonof a cupredoxin to the structural equivalent may be less than about10⁻³, less than about 10⁻⁵, or less than about 10⁻⁷. In otherembodiments, significant structural homology between a cupredoxin andthe structural equivalent may be determined by using the DALI algorithm.Holm & Sander, J. Mol. Biol. 233:123-138 (1993). In specificembodiments, the DALI Z score for a pairwise structural comparison is atleast about 3.5, at least about 7.0, or at least about 10.0.

It is contemplated that the peptides of the composition of invention maybe more than one of a variant, derivative and/or structural equivalentof a cupredoxin. For example, the peptides may be a truncation of azurinthat has been PEGylated, thus making it both a variant and a derivative.In one embodiment, the peptides of the invention are synthesized withα,α-disubstituted non-natural amino acids containing olefin-bearingtethers, followed by an all-hydrocarbon “staple” by ruthenium catalyzedolefin metathesis. Scharmeister et al., J. Am. Chem. Soc. 122:5891-5892(2000); Walensky et al., Science 305:1466-1470 (2004). Additionally,peptides that are structural equivalents of azurin may be fused to otherpeptides, thus making a peptide that is both a structural equivalent anda derivative. These examples are merely to illustrate and not to limitthe invention. Variants, derivatives or structural equivalents ofcupredoxin may or may not bind copper.

In some embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof has some of the pharmacologic activitiesof the P. aeruginosa azurin, and specifically p28. In a specificembodiment, the cupredoxins and variants, derivatives and structuralequivalents of cupredoxins that may inhibit prevent the development ofpremalignant lesions in mammalian cells, tissues or animals, andspecifically but not limited to, mammary gland cells. The invention alsoprovides for the cupredoxins and variants, derivatives and structuralequivalents of cupredoxins that may have the ability to inhibit thedevelopment of mammalian premalignant lesions, and specifically but notlimited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung,colorectal, neck and head, bladder, prostate, skin and cervical cancercells. Inhibition of the development of cancer cells is any decrease, orlessening of the rate of increase, of the development of premalignantlesions that is statistically significant as compared to controltreatments.

Because it is now known that cupredoxins can inhibit the development ofpremalignant lesions and ultimately cancer in mammalian cells, tissuesor animals, and specifically breast cells, and more specifically, mousemammary gland cells, it is now possible to design variants andderivatives of cupredoxins that retain this chemopreventive activity.Such variants, derivatives and structural equivalents can be made by,for example, creating a “library” of various variants, derivatives andstructural equivalents of cupredoxins and cupredoxin derived peptidesand then testing each for chemopreventive activity, and specificallychemopreventive activity in the mouse mammary gland organ culture usingone of many methods known in the art, such the exemplary method inExample 1. It is contemplated that the resulting variants, derivativesand structural equivalents of cupredoxins with chemopreventive activitymay be used in the methods of the invention, in place of or in additionto azurin or p28.

In some specific embodiments, the variant, derivative or structuralequivalent of cupredoxin may inhibit the development of7,12-dimethylbenz (a) anthracene (DMBA) induced premalignant lesions ina mouse mammary gland organ culture (MMOC) to a degree that isstatistically different from a non-treated control. A peptide can betested for this activity by using the MMOC model system is described inExample 1, or as in Mehta et al. (J Natl Cancer Inst 93:1103-1106(2001)) and Mehta et al. (Meth Cell Sci 19:19-24 (1997)). Other methodsto determine whether cancer development is inhibited another are wellknown in the art and may be used as well.

In some specific embodiments, the variant, derivative or structuralequivalent of cupredoxin inhibits the development of mammary alveolarlesions (MAL) in the a MMOC model to a degree that is statisticallydifferent from a non-treated control. In some specific embodiments, thevariant, derivative or structural equivalent of cupredoxin inhibits thedevelopment of mammary ductal lesions (MDL) in the a MMOC model to adegree that is statistically different from a non-treated control. Apeptide can be tested for these activities by using the MMOC modelsystem induced to form premalignant lesions by DMBA, as described inExample 1. Evaluation of development of premalignant lesions in a MMOCmodel system may be determined by morphometic analysis, orhistopathological analysis, as provided in Example 1.

Cupredoxins

These small blue copper proteins (cupredoxins) are electron transferproteins (10-20 kDa) that participate in bacterial electron transferchains or are of unknown function. The copper ion is solely bound by theprotein matrix. A special distorted trigonal planar arrangement to twohistidine and one cysteine ligands around the copper gives rise to verypeculiar electronic properties of the metal site and an intense bluecolor. A number of cupredoxins have been crystallographicallycharacterized at medium to high resolution.

The cupredoxins in general have a low sequence homology but highstructural homology. Gough & Clothia, Structure 12:917-925 (2004); DeRienzo et al., Protein Science 9:1439-1454 (2000). For example, theamino acid sequence of azurin is 31% identical to that of auracyanin B,16.3% to that of rusticyanin, 20.3% to that of plastocyanin, and 17.3%to that of pseudoazurin. See, Table 1. However, the structuralsimilarity of these proteins is more pronounced. The VAST p value forthe comparison of the structure of azurin to auracyanin B is 10^(−7.4),azurin to rusticyanin is 10⁻⁵, azurin to plastocyanin is 10^(−5.6), andazurin to psuedoazurin is 10^(−4.1).

All of the cupredoxins possess an eight-stranded Greek key beta-barrelor beta-sandwich fold and have a highly conserved site architecture. DeRienzo et al., Protein Science 9:1439-1454 (2000). A prominenthydrophobic patch, due to the presence of many long chain aliphaticresidues such as methionines and leucines, is present around the coppersite in azurins, amicyanins, cyanobacterial plastocyanins, cucumberbasic protein and to a lesser extent, pseudoazurin and eukaryoticplastocyanins Id. Hydrophobic patches are also found to a lesser extentin stellacyanin and rusticyanin copper sites, but have differentfeatures. Id.

TABLE 1 Sequence and structure alignment of azurin (1JZG) from P.aeruginosa to other proteins using VAST algorithm. Alignment % aa PDBlength¹ identity P-value² Score³ ^((i)) RMSD⁴ (ii) Description 1AOZ A 282 18.3 10e−7  12.2 1.9 Ascorbate oxidase 1QHQ_A 113 31 10e−7.4 12.1 1)1.9 2) AuracyaninB 1V54 B 1 79 20.3 10e−6.0 11.2 2.1 Cytocrome c oxidase1GY2 A 92 16.3 10e−5.0 11.1 3) 1.8 4) Rusticyanin 3MSP A 74 8.1 10e−6.710.9 2.5 Motile Major Sperm Protein⁵ 1IUZ 74 20.3 10e−5.6 10.3 5) 2.3 6)Plastocyanin 1KGY E 90 5.6 10e−4.6 10.1 7) 3.4 8) Ephrinb2 1PMY 75 17.310e−4.1 9.8 9) 2.3 10) Pseudoazurin ¹Aligned Length: The number ofequivalent pairs of C-alpha atoms superimposed between the twostructures, i.e. how many residues have been used to calculate the 3Dsuperposition. ²P-VAL: The VAST p value is a measure of the significanceof the comparison, expressed as a probability. For example, if the pvalue is 0.001, then the odds are 1000 to 1 against seeing a match ofthis quality by pure chance. The p value from VAST is adjusted for theeffects of multiple comparisons using the assumption that there are 500independent and unrelated types of domains in the MMDB database. The pvalue shown thus corresponds to the p value for the pairwise comparisonof each domain pair, divided by 500. ³Score: The VASTstructure-similarity score. This number is related to the number ofsecondary structure elements superimposed and the quality of thatsuperposition. Higher VAST scores correlate with higher similarity.⁴RMSD: The root mean square superposition residual in Angstroms. Thisnumber is calculated after optimal superposition of two structures, asthe square root of the mean square distances between equivalent C-alphaatoms. Note that the RMSD value scales with the extent of the structuralalignments and that this size must be taken into consideration whenusing RMSD as a descriptor of overall structural similarity. ⁵ C.elegans major sperm protein proved to be an ephrin antagonist in oocytematuration. Kuwabara, Genes and Development 17: 155-161 (2003).

Azurin

The azurins are copper containing proteins of 128 amino acid residueswhich belong to the family of cupredoxins involved in electron transferin certain bacteria. The azurins include those from P. aeruginosa (PA)(SEQ ID NO: 1), A. xylosoxidans, and A. denitrificans. Murphy et al., J.Mol. Biol. 315:859-871 (2002). The amino acid sequence identity betweenthe azurins varies between 60-90%, these proteins showed a strongstructural homology. All azurins have a characteristic β-sandwich withGreek key motif and the single copper atom is always placed at the sameregion of the protein. In addition, azurins possess an essentiallyneutral hydrophobic patch surrounding the copper site. Id.

Plastocyanins

The plastocyanins are soluble proteins of cyanobacteria, algae andplants that contain one molecule of copper per molecule and are blue intheir oxidized form. They occur in the chloroplast, where they functionas electron carriers. Since the determination of the structure of poplarplastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha,Chlamydomonas) and plant (French bean) plastocyanins has been determinedeither by crystallographic or NMR methods, and the poplar structure hasbeen refined to 1.33 Å resolution. SEQ ID NO: 3 shows the amino acidsequence of plastocyanin from Phormidium laminosum, a thermophiliccyanobacterium. Another plastocyanin of interest is from Ulva pertussis.

Despite the sequence divergence among plastocyanins of algae andvascular plants (e.g., 62% sequence identity between the Chlamydomonasand poplar proteins), the three-dimensional structures are conserved(e.g., 0.76 Å rms deviation in the C alpha positions between theChlamydomonas and Poplar proteins). Structural features include adistorted tetrahedral copper binding site at one end of aneight-stranded antiparallel beta-barrel, a pronounced negative patch,and a flat hydrophobic surface. The copper site is optimized for itselectron transfer function, and the negative and hydrophobic patches areproposed to be involved in recognition of physiological reactionpartners. Chemical modification, cross-linking, and site-directedmutagenesis experiments have confirmed the importance of the negativeand hydrophobic patches in binding interactions with cytochrome f, andvalidated the model of two functionally significant electron transferpaths involving plastocyanin. One putative electron transfer path isrelatively short (approximately 4 Å) and involves the solvent-exposedcopper ligand His-87 in the hydrophobic patch, while the other is morelengthy (approximately 12-15 Å) and involves the nearly conservedresidue Tyr-83 in the negative patch. Redinbo et al., J. Bioenerg.Biomembr. 26:49-66 (1994).

Rusticyanins

Rusticyanins are blue-copper containing single-chain polypeptidesobtained from a Thiobacillus (now called Acidithiobacillus). The X-raycrystal structure of the oxidized form of the extremely stable andhighly oxidizing cupredoxin rusticyanin from Thiobacillus ferrooxidans(SEQ ID NO: 4) has been determined by multiwavelength anomalousdiffraction and refined to 1.9 Å resolution. The rusticyanins arecomposed of a core beta-sandwich fold composed of a six- and aseven-stranded b-sheet. Like other cupredoxins, the copper ion iscoordinated by a cluster of four conserved residues (His 85, Cys138,His143, Met148) arranged in a distorted tetrahedron. Walter, R. L. etal., J. Mol. Biol. 263:730-51 (1996).

Pseudoazurins

The pseudoazurins are a family of blue-copper containing single-chainpolypeptide. The amino acid sequence of pseudoazurin obtained fromAchromobacter cycloclastes is shown in SEQ ID NO: 5. The X-ray structureanalysis of pseudoazurin shows that it has a similar structure to theazurins although there is low sequence homology between these proteins.Two main differences exist between the overall structure of thepseudoazurins and azurins. There is a carboxy terminus extension in thepseudoazurins, relative to the azurins, consisting of two alpha-helices.In the mid-peptide region azurins contain an extended loop, shortened inthe pseudoazurins, which forms a flap containing a short α-helix. Theonly major differences at the copper atom site are the conformation ofthe MET side-chain and the Met-S copper bond length, which issignificantly shorter in pseudoazurin than in azurin.

Phytocyanins

The proteins identifiable as phytocyanins include, but are not limitedto, cucumber basic protein, stellacyanin, mavicyanin, umecyanin, acucumber peeling cupredoxin, a putative blue copper protein in pea pods,and a blue copper protein from Arabidopsis thaliana. In all exceptcucumber basic protein and the pea-pod protein, the axial methionineligand normally found at blue copper sites is replaced by glutamine.

Auracyanin

Three small blue copper proteins designated auracyanin A, auracyaninB-1, and auracyanin B-2 have been isolated from the thermophilic greengliding photosynthetic bacterium Chloroflexus aurantiacus. The two Bforms are glycoproteins and have almost identical properties to eachother, but are distinct from the A form. The sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrates apparent monomermolecular masses as 14 (A), 18 (B-2), and 22 (B-1) kDa.

The amino acid sequence of auracyanin A has been determined and showedauracyanin A to be a polypeptide of 139 residues. Van Dreissche et al.,Protein Science 8:947-957 (1999). His58, Cys123, His128, and Met132 arespaced in a way to be expected if they are the evolutionary conservedmetal ligands as in the known small copper proteins plastocyanin andazurin. Secondary structure prediction also indicates that auracyaninhas a general beta-barrel structure similar to that of azurin fromPseudomonas aeruginosa and plastocyanin from poplar leaves. However,auracyanin appears to have sequence characteristics of both small copperprotein sequence classes. The overall similarity with a consensussequence of azurin is roughly the same as that with a consensus sequenceof plastocyanin, namely 30.5%. The N-terminal sequence region 1-18 ofauracyanin is remarkably rich in glycine and hydroxy amino acids. Id.See exemplary amino acid sequence SEQ ID NO: 15 for chain A ofauracyanin from Chloroflexus aurantiacus (NCBI Protein Data BankAccession No. AAM12874).

The auracyanin B molecule has a standard cupredoxin fold. The crystalstructure of auracyanin B from Chloroflexus aurantiacus has beenstudied. Bond et al., J. Mol. Biol. 306:47-67 (2001). With the exceptionof an additional N-terminal strand, the molecule is very similar to thatof the bacterial cupredoxin, azurin. As in other cupredoxins, one of theCu ligands lies on strand 4 of the polypeptide, and the other three liealong a large loop between strands 7 and 8. The Cu site geometry isdiscussed with reference to the amino acid spacing between the latterthree ligands. The crystallographically characterized Cu-binding domainof auracyanin B is probably tethered to the periplasmic side of thecytoplasmic membrane by an N-terminal tail that exhibits significantsequence identity with known tethers in several othermembrane-associated electron-transfer proteins. The amino acid sequencesof the B forms are presented in McManus et al. J. Biol. Chem.267:6531-6540 (1992). See exemplary amino acid sequence SEQ ID NO: 16for chain B of auracyanin from Chloroflexus aurantiacus (NCBI ProteinData Bank Accession No. 1QHQA).

Stellacyanin

Stellacyanins are a subclass of phytocyanins, a ubiquitous family ofplant cupredoxins. An exemplary sequence of a stellacyanin is includedherein as SEQ ID NO: 14. The crystal structure of umecyanin, astellacyanin from horseradish root (Koch et al., J. Am. Chem. Soc.127:158-166 (2005)) and cucumber stellacyanin (Hart et al., ProteinScience 5:2175-2183 (1996)) is also known. The protein has an overallfold similar to the other phytocyanins. The ephrin B2 protein ectodomaintertiary structure bears a significant similarity to stellacyanin. Tothet al., Developmental Cell 1:83-92 (2001). An exemplary amino acidsequence of a stellacyanin is found in the National Center forBiotechnology Information Protein Data Bank as Accession No. 1JER, SEQID NO: 14.

Cucumber Basic Protein

An exemplary amino acid sequence from a cucumber basic protein isincluded herein as SEQ ID NO: 17. The crystal structure of the cucumberbasic protein (CBP), a type 1 blue copper protein, has been refined at1.8 Å resolution. The molecule resembles other blue copper proteins inhaving a Greek key beta-barrel structure, except that the barrel is openon one side and is better described as a “beta-sandwich” or “beta-taco”.Guss et al., J. Mol. Biol. 262:686-705 (1996). The ephrinB2 proteinectodomian tertiary structure bears a high similarity (rms deviation 1.5Å for the 50α carbons) to the cucumber basic protein. Toth et al.,Developmental Cell 1:83-92 (2001).

The Cu atom has the normal blue copper NNSS' co-ordination with bondlengths Cu—N(His39)=1.93 A, Cu—S(Cys79)=2.16 A, Cu—N(His84)=1.95 A,Cu—S(Met89)=2.61 A. A disulphide link, (Cys52)-S—S-(Cys85), appears toplay an important role in stabilizing the molecular structure. Thepolypeptide fold is typical of a sub-family of blue copper proteins(phytocyanins) as well as a non-metalloprotein, ragweed allergen Ra3,with which CBP has a high degree of sequence identity. The proteinscurrently identifiable as phytocyanins are CBP, stellacyanin,mavicyanin, umecyanin, a cucumber peeling cupredoxin, a putative bluecopper protein in pea pods, and a blue copper protein from Arabidopsisthaliana. In all except CBP and the pea-pod protein, the axialmethionine ligand normally found at blue copper sites is replaced byglutamine. An exemplary sequence for cucumber basic protein is found inNCBI Protein Data Bank Accession No. 2CBP, SEQ ID NO: 17.

Methods of Use

The invention provides methods to prevent de novo malignancies inotherwise healthy patients comprising administering to the patient atleast one peptide that is a cupredoxin, or variant, derivative orstructural equivalent thereof, as described above. Chemopreventivetherapies are based on the hypothesis that the interruption of processesinvolved in cancergenesis will prevent the development of cancer. Thecupredoxin Pseudomonas aeruginosa azurin and the truncated azurinpeptide p28 are now known to inhibit the development of premalignantlesions, either by inhibiting the initial formation of premalignantlesions, or killing or inhibiting the growth of premalignant lesionsthat are present. It therefore contemplated that a cupredoxin, orvariant, derivative or structural equivalent thereof, as describedabove, with the ability to inhibit the development of premalignantlesions, may be used in chemopreventive therapies in otherwise healthypatients. Such otherwise healthy patients are, in some embodiments,patients at a higher risk to develop cancer than those in the generalpopulation. Cancers that may be prevented by treatment with thecompositions of the invention include, but are not limited to, melanoma,breast, pancreas, glioblastoma, astrocytoma, lung, colorectal, neck andhead, bladder, prostate, skin, and cervical cancer. In some embodiments,the patient may be human. In other embodiments, the patient is nothuman.

The invention further includes methods to study the development ofcancer comprising contacting mammalian cells before or after inductionwith a carcinogen with a composition comprising cupredoxin, or avariant, derivative or structural equivalent thereof and observing thedevelopment of the cells. In some embodiments, the cells are mousemammary gland cells, while in others they are other cells that maybecome malignant in mammals.

Patients at a higher at risk to develop cancer than the generalpopulation may be patients with high risk features, patients withpremalignant lesions, and patients that have been cured of their initialcancer or definitively treated for their premalignant lesions. Seegenerally Tsao et al., CA Cancer J Clin 54:150-180 (2004). High riskfeatures may be behavioral, genetic, environmental or physiologicalfactors of the patient. Behavioral factors that predispose a patient tovarious forms of cancer include, but are not limited to, smoking, diet,alcohol consumption, hormone replacement therapy, higher body massindex, nulliparity, beta1 nut use, frequent mouthwash use, exposure tohuman papillomavirus, childhood and chronic sun exposure, early age offirst intercourse, multiple sexual partners, and oral contraceptive use.Genetic factors that predispose a patient to various forms of cancerinclude, but are not limited to, a family history of cancer, genecarrier status of BRCA/and BRCA2, prior history of breast neoplasia,familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectalcancer (HNPCC), red or blond hair and fair-skinned phenotype, xerodermapigmentosum, and ethnicity. Environmental features that predispose apatient to various forms of cancer include, but are not limited to,exposure to radon, polycyclic aromatic hydrocarbons, nickel, chromate,arsenic, asbestos, chloromethyl ethers, benzo[a]pyrene, radiation, andaromatic amines from rubber or paint occupational exposure. Othermiscellaneous factors that predispose a patient to various forms ofcancer include, but are not limited to, chronic obstructive pulmonarydisease with airflow obstruction, chronic bladder infections,schistosomiasis, older age, and immunocompromised status.

Additionally, patients at a higher risk of developing cancer may bedetermined by the use of various risk models that have been developedfor certain kinds of cancer. For example, patients predisposed to breastcancer may be determined using the Gail risk model, or the Claus model,among others. See Gail et al., J Natl Cancer Inst 81:1879-1886 (1989);Cuzick, Breast 12:405-411 (2003); Huang et al., Am J Epidemiol151:703-714 (2000).

Patients with premalignant lesions are at a higher risk to developcancer than the general population. The presence of premalignant lesionsin or on a patient may be determined by many methods that are well knownto those in the art. Intermediate markers or biomarkers that originatefrom premalignant lesions may be measured in a patient to determine ifthe patient harbors premalignant lesions. Chromosomal abnormalitiesoccur in tumor cells and the adjacent histologicially normal tissues inthe majority of cancer patients. Progression in chromosomalabnormalities parallels the phenotypic progression from premalignantlesion to invasive cancer. Thiberville et al., Cancer Res. 55:5133-5139(1995). Therefore, chromosomal abnormalities associated with cancer maybe used as intermediate markers to detect premalignant lesions in apatient. Common chromosomal abnormalities associated with cancerinclude, but are not limited to, allelic deletions or loss ofheterozygosity (LOH) in tumor suppressor genes such as 3p (FHIT andothers), 9p (9p21 for p16^(INK4), p15^(INK4B), and p19^(ARF)), 17p(17p13 for p53 gene and others) and 13q (13q14 for retinoblastoma geneRb and others). Deletions in 3p and 9p are associated with smoking andthe early stages of lung cancer. Mao et al., J. Natl. Cancer Inst.89:857-862 (1997). Deletions affecting 3p, 5q, 8p, 17p and 18q arecommon change in epithelial cancers. See generally Tsao et al., CA Clin.Cancer J. Clin. 54:153 (2004). Other chromosomal mutations associatedwith cancer include those which activate oncogenes. Oncogenes whosepresence may be used as intermediate markers include, but are notlimited to, Ras, c-myc, epidemral growth factor, erb-B2 and cyclins E,D1 and B1. See generally id. at 154.

Other intermediate markers may be the products of genes up-regulated inpremalignant cells and cancer cells. Genes that may be up-regulated inpremalignant cells include, but are not limited to, cyclooxygenasesCOX-1 and COX-2, telomerase. Other biomarkers of cancer cells, and somepremalignant cells, include, but are not limited to, p53, epidermalgrowth factor receptor (GFR), proliferating cell nuclear antigen (PCNA),RAS, COX-2, Ki-67, DNA aneuploidy, DNA polymerase-α, ER, Her2neu,E-cadherin, RARβ, hTERT, p16^(INK4a), FHIT (3p14), Bc1-2, VEGF-R, HPVinfection, LOH 9p21, LOH 17p, p-AKT, hnRNP A2/B1, RAF, Myc, c-KIT,cyclin D1, E and B1, IGF1, bc1-2, p16, LOH 3p21.3, LOH 3p2.5, LOH 9p21,LOH 17p13, LOH 13q, LOH 8p, hMSH2, APC, DCC, DPC4, JV18, BAX, PSA,GSTP1, NF-kB, AP1, D3S2, HPV infection, LOH 3p14, LOH 4q, LOH 0.5p,bladder tumor antigen (BTA), BTK TRAK (Alidex, Inc., Redmond Wash.),urinary tract matrix protein 22, fibrin degradation product, autodrinemotility factor receptor, BCLA-4, cytokeratin 20, hyaluronic acid, CYFRA21-1, BCA, beta-human chorionic gonadotropin, and tissue polypeptideantigen (TPA). See generally id. at 155-157.

Patients that have been cured of their initial cancers or have beendefinitively treated for their premalignant lesions are also at a higherrisk to develop cancer than the general population. A second primarytumor refers to a new primary cancer in a person with a history ofcancer. Second primary tumors are the leading cause of mortality in headand neck cancer. Id. at 150. A second primary tumor is distinct from ametastasis in that the former originates de novo while the lateroriginates from an existing tumor. Patients that have been cured ofcancer or premalignant lesions of the breast, head and neck, lung, andskin are at a particularly high risk to develop second primary tumors.

The compositions comprising a cupredoxin or variant, derivative orstructural equivalent thereof can be administered to the patient by manyroutes and in many regimens that will be well known to those in the art.In specific embodiments, the cupredoxin, or variant, derivative orstructural equivalent thereof is administered intravenously,intramuscularly, subcutaneously, topically, orally, or by inhalation.The compositions may be administered to the patient by any means thatdelivers the peptides to the site in the patient that is at risk ofdeveloping cancer. In specific embodiments, the cupredoxin or variant,derivative or structural equivalent thereof is administeredintraveneously.

In one embodiment, the methods may comprise co-administering to apatient one unit dose of a composition comprising a cupredoxin or avariant, derivative or structural equivalent of cupredoxin and one unitdose of a composition comprising another chemopreventive drug, in eitherorder, administered at about the same time, or within about a given timefollowing the administration of the other, for example, about one minuteto about 6o minutes following the administration of the other drug, orabout 1 hour to about 12 hours following the administration of the otherdrug. Chemopreventive drugs of interest include, but are not limited to,tamoxifen, aromatase inhibitors such as letrozole and anastrozole(Arimidex®), retinoids such as N-[4-hydroxyphenyl]retinamide (4-HPR,fenretinide), nonsteriodal antiinflammatory agents (NSAIDs) such asaspirin and sulindac, celecoxib (COX-2 inhibitor),defluoromethylornithing (DFMO), ursodeoxycholic acid,3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, EKI-785(EGFR inhibitor), bevacizumab (antibody to VEGF-receptor), cetuximab(antibody to EGFR), retinol such as vitamin A, beta-carotene, 13-cisretinoic acid, isotretinoin and retinyl palmitate, α-tocopherol,interferon, oncolytic adenovirus d11520 (ONYX-015), gefitinib,etretinate, finasteride, indole-3-carbinol, resveratrol, chlorogenicacid, raloxifene, and oltipraz.

Pharmaceutical Compositions Comprising Cupredoxin, or Variant,Derivative or Structural Equivalent Thereof

Pharmaceutical compositions comprising cupredoxin or variant, derivativeor structural equivalents thereof, can be manufactured in anyconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. The substantially pure or pharmaceutical gradecupredoxin or variants, derivatives and structural equivalents thereofcan be readily combined with a pharmaceutically acceptable carrierwell-known in the art. Such carriers enable the preparation to beformulated as a tablet, pill, dragee, capsule, liquid, gel, syrup,slurry, suspension, and the like. Suitable carriers or excipients canalso include, for example, fillers and cellulose preparations. Otherexcipients can include, for example, flavoring agents, coloring agents,detackifiers, thickeners, and other acceptable additives, adjuvants, orbinders. In some embodiments, the pharmaceutical preparation issubstantially free of preservatives. In other embodiments, thepharmaceutical preparation may contain at least one preservative.General methodology on pharmaceutical dosage forms is found in Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems (LippencottWilliams & Wilkins, Baltimore Md. (1999)).

The composition comprising a cupredoxin or variant, derivative orstructural equivalent thereof used in the invention may be administeredin a variety of ways, including by injection (e.g., intradermal,subcutaneous, intramuscular, intraperitoneal and the like), byinhalation, by topical administration, by suppository, by using atransdermal patch or by mouth. General information on drug deliverysystems can be found in Ansel et al., id. In some embodiments, thecomposition comprising a cupredoxin or variant, derivative or structuralequivalent thereof can be formulated and used directly as injectibles,for subcutaneous and intravenous injection, among others. The injectableformulation, in particular, can advantageously be used to treat patientsthat are appropriate for chemopreventive therapy. The compositioncomprising a cupredoxin or variant, derivative or structural equivalentthereof can also be taken orally after mixing with protective agentssuch as polypropylene glycols or similar coating agents.

When administration is by injection, the cupredoxin or variant,derivative or structural equivalent thereof may be formulated in aqueoussolutions, specifically in physiologically compatible buffers such asHanks solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the cupredoxin or variant,derivative or structural equivalent thereof may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. In some embodiments, the pharmaceutical composition does notcomprise an adjuvant or any other substance added to enhance the immuneresponse stimulated by the peptide. In some embodiments, thepharmaceutical composition comprises a substance that inhibits an immuneresponse to the peptide.

When administration is by intravenous fluids, the intravenous fluids foruse administering the cupredoxin or variant, derivative or structuralequivalent thereof may be composed of crystalloids or colloids.Crystalloids as used herein are aqueous solutions of mineral salts orother water-soluble molecules. Colloids as used herein contain largerinsoluble molecules, such as gelatin. Intravenous fluids may be sterile.

Crystalloid fluids that may be used for intravenous administrationinclude but are not limited to, normal saline (a solution of sodiumchloride at 0.9% concentration), Ringer's lactate or Ringer's solution,and a solution of 5% dextrose in water sometimes called D5W, asdescribed in Table 2.

TABLE 2 Composition of Common Crystalloid Solutions Solution Other Name[Na⁺] [Cl⁻] [Glucose] D5W 5% Dextrose 0 0 252 2/3 & 1/3 3.3% Dextrose/51 51 168 0.3% saline Half-normal 0.45% NaCl 77 77 0 saline Normalsaline 0.9% NaCl 154 154 0 Ringer's Ringer's 130 109 0 lactate* solution*Ringer's lactate also has 28 mmol/L lactate, 4 mmol/L K⁺ and 3 mmol/LCa²⁺.

When administration is by inhalation, the cupredoxin or variant,derivative or structural equivalent thereof may be delivered in the formof an aerosol spray from pressurized packs or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the proteins and a suitable powderbase such as lactose or starch.

When administration is by topical administration, the cupredoxin orvariant, derivative or structural equivalent thereof may be formulatedas solutions, gels, ointments, creams, jellies, suspensions, and thelike, as are well known in the art. In some embodiments, administrationis by means of a transdermal patch. When administration is bysuppository (e.g., rectal or vaginal), cupredoxin or variants andderivatives thereof compositions may also be formulated in compositionscontaining conventional suppository bases.

When administration is oral, a cupredoxin or variant, derivative orstructural equivalent thereof can be readily formulated by combining thecupredoxin or variant, derivative or structural equivalent thereof withpharmaceutically acceptable carriers well known in the art. A solidcarrier, such as mannitol, lactose, magnesium stearate, and the like maybe employed; such carriers enable the cupredoxin and variants,derivatives or structural equivalent thereof to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.For oral solid formulations such as, for example, powders, capsules andtablets, suitable excipients include fillers such as sugars, cellulosepreparation, granulating agents, and binding agents.

Other convenient carriers, as well-known in the art, also includemultivalent carriers, such as bacterial capsular polysaccharide, adextran or a genetically engineered vector. In addition,sustained-release formulations that include a cupredoxin or variant,derivative or structural equivalent thereof allow for the release ofcupredoxin or variant, derivative or structural equivalent thereof overextended periods of time, such that without the sustained releaseformulation, the cupredoxin or variant, derivative or structuralequivalent thereof would be cleared from a subject's system, and/ordegraded by, for example, proteases and simple hydrolysis beforeeliciting or enhancing a therapeutic effect.

The half-life in the bloodstream of the peptides of the invention can beextended or optimized by several methods well known to those in the art.The peptide variants of the invention may include, but are not limitedto, various variants that may increase their stability, specificactivity, longevity in the bloodstream, and/or decrease immunogenicityof the cupredoxin, while retaining the ability of the peptide to inhibitthe development of premalignant lesions in mammalian cells, tissues andanimals. Such variants include, but are not limited to, those whichdecrease the hydrolysis of the peptide, decrease the deamidation of thepeptide, decrease the oxidation, decrease the immunogenicity, increasethe structural stability of the peptide or increase the size of thepeptide. Such peptides also include circularized peptides (see Monk etal., BioDrugs 19(4):261-78, (2005); DeFreest et al., J. Pept. Res.63(5):409-19 (2004)), D,L-peptides (diastereomer), Futaki et al., J.Biol. Chem. February 23; 276(8):5836-40 (2001); Papo et al., Cancer Res.64(16):5779-86 (2004); Miller et al., Biochem. Pharmacol. 36(1):169-76,(1987)); peptides containing unusual amino acids (see Lee et al., J.Pept. Res. 63(2):69-84 (2004)), N- and C-terminal modifications (seeLabrie et al., Clin. Invest. Med. 13(5):275-8, (1990)), hydrocarbonstapling (see Schafineister et al., J. Am. Chem. Soc. 122:5891-5892(2000); Walenski et al., Science 305:1466-1470 (2004)) and PEGylation.

In various embodiments, the pharmaceutical composition includes carriersand excipients (including but not limited to buffers, carbohydrates,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), water, oils, saline solutions,aqueous dextrose and glycerol solutions, other pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like. It will be recognized that, while any suitablecarrier known to those of ordinary skill in the art may be employed toadminister the compositions of this invention, the type of carrier willvary depending on the mode of administration. Compounds may also beencapsulated within liposomes using well-known technology. Biodegradablemicrospheres may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268; 5,075,109;5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.

The pharmaceutical compositions may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration.

Administration of Cupredoxin or Variant, Derivative or StructuralEquivalent Thereof

The cupredoxin or variant, derivative or structural equivalent thereofcan be administered formulated as pharmaceutical compositions andadministered by any suitable route, for example, by oral, buccal,inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical,percutaneous, i.e., transdermal or parenteral (including intravenous,intramuscular, subcutaneous and intracoronary) or vitreousadministration. The pharmaceutical formulations thereof can beadministered in any amount effective to achieve its intended purpose.More specifically, the composition is administered in a therapeuticallyeffective amount. In specific embodiments, the therapeutically effectiveamount is generally from about 0.01-20 mg/day/kg of body weight.

The compounds comprising cupredoxin or variant, derivative or structuralequivalent thereof are useful for the prevention of cancer, alone or incombination with other active agents. The appropriate dosage will, ofcourse, vary depending upon, for example, the compound of cupredoxin orvariant, derivative or structural equivalent thereof employed, the host,the mode of administration and the nature and severity of the potentialcancer. However, in general, satisfactory results in humans areindicated to be obtained at daily dosages from about 0.01-20 mg/kg ofbody weight. An indicated daily dosage in humans is in the range fromabout 0.7 mg to about 1400 mg of a compound of cupredoxin or variant,derivative or structural equivalent thereof conveniently administered,for example, in daily doses, weekly doses, monthly doses, and/orcontinuous dosing. Daily doses can be in discrete dosages from 1 to 12times per day. Alternatively, doses can be administered every other day,every third day, every fourth day, every fifth day, every sixth day,every week, and similarly in day increments up to 31 days or over.Alternatively, dosing can be continuous using patches, i.v.administration and the like.

The exact formulation, route of administration, and dosage is determinedby the attending physician in view of the patient's condition. Dosageamount and interval can be adjusted individually to provide plasmalevels of the active cupredoxin or variant, derivative or structuralequivalent thereof which are sufficient to maintain therapeutic effect.Generally, the desired cupredoxin or variant, derivative or structuralequivalent thereof is administered in an admixture with a pharmaceuticalcarrier selected with regard to the intended route of administration andstandard pharmaceutical practice.

In one aspect, the cupredoxin or variant, derivative or structuralequivalent thereof is delivered as DNA such that the polypeptide isgenerated in situ. In one embodiment, the DNA is “naked,” as described,for example, in Ulmer et al., (Science 259:1745-1749 (1993)) andreviewed by Cohen (Science 259:1691-1692 (1993)). The uptake of nakedDNA may be increased by coating the DNA onto a carrier, e.g.,biodegradable beads, which are then efficiently transported into thecells. In such methods, the DNA may be present within any of a varietyof delivery systems known to those of ordinary skill in the art,including nucleic acid expression systems, bacterial and viralexpression systems. Techniques for incorporating DNA into suchexpression systems are well known to those of ordinary skill in the art.See, e.g., WO90/11092, WO93/24640, WO 93/17706, and U.S. Pat. No.5,736,524.

Vectors, used to shuttle genetic material from organism to organism, canbe divided into two general classes: Cloning vectors are replicatingplasmid or phage with regions that are essential for propagation in anappropriate host cell and into which foreign DNA can be inserted; theforeign DNA is replicated and propagated as if it were a component ofthe vector. An expression vector (such as a plasmid, yeast, or animalvirus genome) is used to introduce foreign genetic material into a hostcell or tissue in order to transcribe and translate the foreign DNA,such as the DNA of a cupredoxin. In expression vectors, the introducedDNA is operably-linked to elements such as promoters that signal to thehost cell to highly transcribe the inserted DNA. Some promoters areexceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Operably-linking acupredoxin and variants and derivatives thereof polynucleotide to aninducible promoter can control the expression of the cupredoxin andvariants and derivatives thereof in response to specific factors.Examples of classic inducible promoters include those that areresponsive to α-interferon, heat shock, heavy metal ions, and steroidssuch as glucocorticoids (Kaufman, Methods Enzymol. 185:487-511 (1990))and tetracycline. Other desirable inducible promoters include those thatare not endogenous to the cells in which the construct is beingintroduced, but, are responsive in those cells when the induction agentis exogenously supplied. In general, useful expression vectors are oftenplasmids. However, other forms of expression vectors, such as viralvectors (e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses) are contemplated.

Vector choice is dictated by the organism or cells being used and thedesired fate of the vector. In general, vectors comprise signalsequences, origins of replication, marker genes, polylinker sites,enhancer elements, promoters, and transcription termination sequences.

Kits Comprising Cupredoxin, or Variant, Derivative or StructuralEquivalent Thereof

In one aspect, the invention provides regimens or kits comprising one ormore of the following in a package or container: (1) a pharmacologicallyactive composition comprising at least one cupredoxin or variant,derivative or structural equivalent thereof; (2) an additionalchemopreventive drug, (3) apparatus to administer the biologicallyactive composition to the patient, such as a syringe, nebulizer etc.

When a kit is supplied, the different components of the composition maybe packaged in separate containers, if appropriate, and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized cupredoxin andvariants, derivatives and structural equivalents thereof, or buffersthat have been packaged under a neutral, non-reacting gas, such asnitrogen. Ampules may consist of any suitable material, such as glass,organic polymers, such as polycarbonate, polystyrene, etc., ceramic,metal or any other material typically employed to hold similar reagents.Other examples of suitable containers include simple bottles that may befabricated from similar substances as ampules, and envelopes, that maycomprise foil-lined interiors, such as aluminum or an alloy. Othercontainers include test tubes, vials, flasks, bottles, syringes, or thelike. Containers may have a sterile access port, such as a bottle havinga stopper that can be pierced by a hypodermic injection needle. Othercontainers may have two compartments that are separated by a readilyremovable membrane that upon removal permits the components to be mixed.Removable membranes may be glass, plastic, rubber, etc.

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audiotape, flash memory device etc. Detailedinstructions may not be physically associated with the kit; instead, auser may be directed to an internet web site specified by themanufacturer or distributor of the kit, or supplied as electronic mail.

Modification of Cupredoxin and Variants, Derivatives and StructuralEquivalents Thereof

Cupredoxin or variant, derivative or structural equivalents thereof maybe chemically modified or genetically altered to produce variants andderivatives as explained above. Such variants and derivatives may besynthesized by standard techniques.

In addition to naturally-occurring allelic variants of cupredoxin,changes can be introduced by mutation into cupredoxin coding sequencethat incur alterations in the amino acid sequences of the encodedcupredoxin that do not significantly alter the ability of cupredoxin toinhibit the development of premalignant lesions. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequences of the cupredoxin without altering pharmacologic activity,whereas an “essential” amino acid residue is required for suchpharmacologic activity. For example, amino acid residues that areconserved among the cupredoxins are predicted to be particularlynon-amenable to alteration, and thus “essential.”

Amino acids for which conservative substitutions that do not change thepharmacologic activity of the polypeptide can be made are well known inthe art. Useful conservative substitutions are shown in Table 3,“Preferred substitutions.” Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the invention so long as the substitution doesnot materially alter the pharmacologic activity of the compound.

TABLE 3 Preferred substitutions Preferred Original residue Exemplarysubstitutions substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln,Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser SerGln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln,Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu Norleucine Leu (L)Norleucine, Ile, Val, Met, Ala, Ile Phe Lys (K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) AlaAla Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp,Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, Leu Norleucine

Non-conservative substitutions that affect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge, (3) hydrophobicity, or (4) the bulk of the side chain of thetarget site can modify the pharmacologic activity. Residues are dividedinto groups based on common side-chain properties as denoted in Table 4.Non-conservative substitutions entail exchanging a member of one ofthese classes for another class. Substitutions may be introduced intoconservative substitution sites or more specifically into non-conservedsites.

TABLE 4 Amino acid classes Class Amino acids hydrophobic Norleucine,Met, Ala, Val, Leu, Ile neutral hydrophilic Cys, Ser, Thr acidic Asp,Glu basic Asn, Gln, His, Lys, Arg disrupt chain conformation Gly, Proaromatic Trp, Tyr, Phe

The variant polypeptides can be made using methods known in the art suchas oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. Site-directed mutagenesis (Carter,Biochem J. 237:1-7 (1986); Zoller and Smith, Methods Enzymol.154:329-350 (1987)), cassette mutagenesis, restriction selectionmutagenesis (Wells et al., Gene 34:315-323 (1985)) or other knowntechniques can be performed on the cloned DNA to produce the cupredoxinvariant DNA.

Known mutations of cupredoxins can also be used to create variantcupredoxin to be used in the methods of the invention. For example, theC112D and M44KM64E mutants of azurin are known to have cytotoxic andgrowth arresting activity that is different from the native azurin, andsuch altered activity can be useful in the treatment methods of thepresent invention.

A more complete understanding of the present invention can be obtainedby reference to the following specific Examples. The Examples aredescribed solely for purposes of illustration and are not intended tolimit the scope of the invention. Changes in form and substitution ofequivalents are contemplated as circumstances may suggest or renderexpedient. Although specific terms have been employed herein, such termsare intended in a descriptive sense and not for purposes of limitations.Modifications and variations of the invention as hereinbefore set forthcan be made without departing from the spirit and scope thereof.

EXAMPLES Example 1 Effect of Peptide P-28 on DMBA-Induced MammaryLesions in the MMOC Model

The mouse mammalary gland organ culture (MMOC) model allows evaluatingefficacy of potentially chemopreventive agents against development ofmammary alveolar lesions (MAL) or mammary ductal lesions (MDL) inresponse to DMBA. DMBA under appropriate incubation conditions formeither MAL or MDL based on the hormonal milieu in the medium. Hawthorneet al., Pharmaceutical Biology 40: 70-74 (2002); Mehta et al., J. Natl.Cancer Inst. 93: 1103-1106 (2001). Estrogen and progesterone-treatedglands in culture develop ductal lesions whereas aldosterone andhydrocortisone-treated glands form estrogen and progesterone-independentalveolar lesions. Mammary glands not exposed to a carcinogen orchemopreventive agent, undergo structural regression in the absence ofgrowth-promoting hormones, whereas treatment with DMBA for the 24-hrperiod between days 3 and 4 prevents the regression of structures causedby deprivation of hormones. It is assumed that this is because theglands have lost normal hormonal responsiveness and now have alteredtheir course of development. Generating mammary adenocarcinoma bytransplanting transformed cells into syngeneic mice has proved thepremalignant preneoplastic nature of these unrepressed areas.

The thoracic pair of mammary glands was excised aseptically from eachBalb/c mouse, and the glands were divided into several groups. Theeffects of p28 were evaluated at 4 different dilutions in the medium.Carcinogen treated glands without the test agent served as a measure todetermine percent incidence in the absence of a chemopreventive agent.An additional control was included to serve as a positive control forchemoprevention. Azurin was included in the medium at 50 μg/mlconcentration. For alveolar lesions (MAL) stained glands were evaluatedfor the incidence of lesions (glands containing any lesions as comparedto total number of glands in a given treatment group). For the ductallesions (MDL) similar protocol was adapted, however, as indicated belowin the methods section the hormonal combination is different foralveolar and ductal lesions. The glands were fixed in formalin and thenprocessed for histopathology. The sections are stained with eosin andhematoxelene and evaluated under microscope. Here the multiplicity ofductal lesions between the control and the treatment groups arecompared.

Organ Culture Procedure.

The experimental animals used for the studies were young, virgin BALB/cfemale mice 3 to 4 weeks of age obtained from Charles River, Wilmington,Mass. The mice were treated daily by subcutaneous injections with 1 μgestradiol-17β+1 mg progesterone for 9 days. This treatment is aprerequisite inasmuch as animals not pretreated with steroids fail torespond to hormones in vitro. The entire culture procedure is describedin detail. Jang et al., Science 275:218-220 (1997); Mehta, Eu. J. Cancer36:1275-1282 (2000); Mehta et al., J. Natl. Cancer Inst. 89:212-219(1997); Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001).

Briefly, the animals were killed by cervical dislocation, and thethoracic pair of mammary glands were dissected out on silk rafts andincubated for 10 days in serum free Waymouth MB752/1 medium (5-glands/5ml/dish). The medium was supplemented with glutamine, antibiotics(penicillin and streptomycin 100 units/ml medium) and growth-promotinghormones, 5 μg insulin (I), 5 μg prolactin (P), 1 μg aldosterone (A) and1 μg hydrocortisone (H) per ml of medium for the protocol to inducemammary alveolar lesions (MAL). For induction of ductal lesions (MDL),the medium contained 5 μg/ml, 5 μg/ml P, 0.001 μg/ml estradiol 17β and 1μg/ml progesterone (Pg). Mehta et al., J. Natl. Cancer Inst.93:1103-1106 (2001). The carcinogen, DMBA (2 μg/ml) was added to themedium between days 3 and 4. For the present study, DMBA was dissolvedin DMSO at a final concentration of 4 mg/ml, and 50 μg I was added to100 ml medium resulting in 2 μg/ml final concentrations. The controldishes contained DMSO as vehicle.

On day 4, DMBA is removed from the medium by rinsing the glands in freshmedium and transferring them to new dishes containing fresh mediumwithout DMBA. After 10 days of incubation, the glands were maintainedfor another 14 days in the medium containing only I (5 μg/ml). Duringthe entire culture period, the glands were maintained at 37° C. under95% O₂ and 5% CO₂ environment. The chemopreventive agent was included inthe medium during the first ten days of growth-promoting phase. The testpeptide p28 was evaluated at 4 concentrations ranging from 12.5 μg/ml to100 μg/ml. Azurin was evaluated at 50 μg/ml in the medium. The peptidewas dissolved in sterile water and filtered prior to use. The medium waschanged three times per week (Monday, Wednesday and Friday). At the endof the exposure, the glands were fixed in formalin.

Results were analyzed by Chi-square analysis and Fisher's Exact Test.

Morphometic Analysis of MAL.

For examination of MAL, the glands were stained in alum carmine, andevaluated for the presence of the lesions. The glands were scored forthe presence or absence of mammary lesions, severity of lesions pergland, and toxicity of the agent. The glands stored in xylene wereevaluated for the presence or absence, incidence, and severity ofmammary lesions for each gland under a dissecting microscope. Mammaryglands were scored as positive or negative for mammary lesions, and thepercent incidence was determined as a ratio of glands exhibiting lesionsand the total number of glands in that group. Dilation of ducts ordisintegration of mammary structure because of treatment withchemopreventive agent was considered a toxic effect. The data weresubjected to statistical analysis for the incidence to determine theeffectiveness of the potential chemopreventive agents.

FIG. 1A shows a representative photograph of alveolar lesions in a DMBAtreated gland and its comparison with a gland that was treated with DMBAalong with a chemopreventive agent. The effects of p28 on thedevelopment of alveolar lesion are shown in FIGS. 1B-1G and summarizedin FIG. 2. The peptide p28 inhibited MAL formation by 67% at 25 μg/mlconcentration. Increasing concentration further up to 100 μg/ml did notenhance the efficacy of the peptide. The comparison of the peptide withazurin indicated that p28 was as effective as azurin for MALdevelopment. Azurin at 50 μg/ml concentration resulted in a 67%inhibition. Statistical analyses indicated that the effect of p28 wasstatistically significant compared to DMBA control at concentrationsgreater than 12.5 μg/ml (p<0.01, Fisher's Exact Test; Chi Squareanalysis).

Histopathological Evaluation of MDL.

For MDL, the glands were processed for histopathological evaluations.The glands were sectioned longitudinally into 5-micron sections andstained with eosin hematoxeline. The longitudinal section of each glandwas divided into several fields and each field was evaluated for ductallesions. Mehta et al., J. Natl. Cancer Inst. 93:1103-1106 (2001).Briefly, the entire gland is evaluated under the scope; smaller glandswill have fewer total fields as compared to larger glands. Thus, eachgland will have variable number of fields. Often the number of sectionsthrough the ducts also varies greatly from gland to gland. This resultsin the variable number from group to group. Fields containing ductalhyperplasia or atypia were determined and were compared with totalnumber of field evaluated for each gland. No discrimination is madebetween the hyperplasia or atypia and severely occluded glands. Anyfield containing any of these histological patterns was consideredpositive for the lesion. The treatment groups were compared with thecontrols for the severity and percent inhibition was calculated.

FIG. 3 shows a representative ductal lesion. DMBA induces ductal lesionsvarying from hyperplasia, atypia to complete occlusion of the ducts. Aratio of ductal lesions/total number of ductal sections was determined.Again, 12.5 μg/ml concentration of p28 suppressed only 15% of the MDLformation. However, at 25 μg/ml there was a significant inhibition ofthe lesions comparable to that observed with 50 μg/ml azurin. Theefficacy of p28 at concentrations greater than 12.5 μg/ml wasstatistically significant (p<0.01, Fishers Exact Test). These resultsare summarized in FIG. 4. Often effects of chemopreventive agents can bedifferentiated between the MAL and MDL. For example tamoxifen inhibitedthe development of MDL but not MAL. It is interesting to note thatazurin and p28 inhibited both estrogen and progesterone-dependent ductallesions as well as independent alveolar lesions.

This example indicates that both p28 and azurin can prevent thedevelopment of precancerous lesions in breast tissue. Thus, p28 andazurin may be used as chemopreventive agents in mammalian patients.

1. An isolated peptide that is a variant, derivative or structuralequivalent of a cupredoxin; and that can inhibit the development ofpremalignant lesions in mammalian tissue.
 2. The isolated peptide ofclaim 1, wherein the cupredoxin is selected from the group consisting ofazurin, pseudoazurin, plastocyanin, rusticyanin, Laz, auracyanin,stellacyanin and cucumber basic protein.
 3. The isolated peptide ofclaim 2, wherein the cupredoxin is azurin.
 4. The isolated peptide ofclaim 1, wherein the cupredoxin is from an organism selected from thegroup consisting of Pseudomonas aeruginosa, Alcaligenes faecalis,Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp.,Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens,Pseudomonas chlororaphis, Xylella fastidiosa and Vibrioparahaemolyticus.
 5. The isolated peptide of claim 4, that is fromPseudomonas aeruginosa.
 6. The isolated peptide of claim 1, which ispart of a peptide selected from the group consisting of SEQ ID NOS: 1,3-19.
 7. The isolated peptide of claim 1, to which a sequence selectedfrom the group consisting of SEQ ID NOS: 1, 3-19 has at least 80% aminoacid sequence identity.
 8. The isolated peptide of claim 1, which is atruncation of the cupredoxin.
 9. The isolated peptide of claim 8,wherein the peptide is more than about 10 residues and not more thanabout 100 residues.
 10. The isolated peptide of claim 8, wherein thepeptide comprises a sequence selected from the group consisting ofPseudomonas aeruginosa azurin residues 50-77, Pseudomonas aeruginosaazurin residues 50-67, Pseudomonas aeruginosa azurin residues 36-88, andSEQ ID NOS: 20-24.
 11. The isolated peptide of claim 10, wherein thepeptide consists of a sequence selected from the group consisting ofPseudomonas aeruginosa azurin residues 50-77, Pseudomonas aeruginosaazurin residues 50-67, Pseudomonas aeruginosa azurin residues 36-88 andSEQ ID NOS: 20-24.
 12. The isolated peptide of claim 1, wherein thepeptide comprises equivalent residues of a cupredoxin as a region ofPseudomonas aeruginosa azurin selected from the group consisting ofresidues 50-77, residues 50-67 and residues 36-88.
 13. A pharmaceuticalcomposition, comprising at least one cupredoxin or peptide of claim 1 ina pharmaceutically acceptable carrier.
 14. The pharmaceuticalcomposition of claim 13 which comprises at least two of the cupredoxinsor peptides.
 15. The composition of claim 13, wherein the pharmaceuticalcomposition is formulated for intravenous administration.
 16. Thecomposition of claim 13, wherein the cupredoxin is from an organismselected from the group consisting of Pseudomonas aeruginosa,Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetellabronchiseptica, Methylomonas sp., Neisseria meningitidis, Neisseriagonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylellafastidiosa and Vibrio parahaemolyticus.
 17. The composition of claim 16,wherein the cupredoxin is from Pseudomonas aeruginosa.
 18. Thecomposition of claim 13, wherein the cupredoxin is selected from thegroup consisting of SEQ ID NOS: 1, 3-19.
 19. A method to treat amammalian patient, comprising administering to the patient atherapeutically effective amount of the composition of claim
 13. 20. Themethod of claim 19, wherein the patient is human.
 21. The method ofclaim 19, wherein the patient is at a higher risk to develop cancer thanthe general population.
 22. The method of claim 21, wherein the canceris selected from melanoma, breast, pancreas, glioblastoma, astrocytoma,lung, colorectal, neck and head, bladder, prostate, skin, and cervicalcancer.
 23. The method of claim 21, wherein the patient has at least onehigh risk feature.
 24. The method of claim 19, wherein the patient haspremalignant lesions.
 25. The method of claim 19, wherein the patienthas been cured of cancer or premalignant lesions.
 26. The method ofclaim 19, wherein the pharmaceutical composition is administered by amode selected from the group consisting of intravenous injection,intramuscular injection, subcutaneous injection, inhalation, topicaladministration, transdermal patch, suppository, vitreous injection andoral.
 27. The method of claim 24, wherein the mode of administration isby intravenous injection.
 28. The method of claim 21, wherein thepharmaceutical composition is co-administered with at least one otherchemopreventive drug.
 29. The method of claim 26, wherein thepharmaceutical composition is administered at about the same time asanother chemopreventive drug.
 30. A kit comprising the composition ofclaim 13 in a vial.
 31. The kit of claim 30, wherein the kit is designedfor intravenous administration.
 32. A method to study the development ofcancer comprising contacting the mammalian cells with a cupredoxin orpeptide of claim 1; and measuring the development of premalignant andmalignant cells.
 33. The method of claim 32, wherein the cells are humancells.
 34. The method of claim 32, wherein the cells are mammary glandcells.
 35. The method of claim 32, wherein the cells are induced todevelop cancer.
 36. An expression vector, which encodes the peptide ofclaim 1.